Dear Erhan

The staining for the nervous system are the most complex .

There are two main types of staining for a silver impregnation , a type is performed on the sample first to cutting to the microtome and the other after cutting.

I 've always preferred the second method , and in particular for the Central Nervous System you should perform staining of Bodian with protargolo silver: this method

1 . Attach Duboscq -Brasil , Bouin , Carnoy etc..

2 . perform the ascending scale up to 100 alcohol

3 . make two infiltration (15 ') in xylene

4 . included in paraffin

5. make sections of 10-12 microns and place them on a microscope slide and run the descending scale of alcohols to the water )

6 . incubate slides with sections for 12-24 hours in a thermostat at 37 ° C in a solution of 1% protargolo + some pieces of metallic copper immersed in this solution

7. lavare in distilled water

8.reazione reduction for 5-10 minutes :

Hydroquinone 1gr .

5g sodium sulfite .

distilled water 100ml

9. wash for 1 minutes in distilled water

10 . 4-5 minutes in a solution of gold chloride (check under a microscope , brown)

11 . 5-10 minutes of sodium thiosulphate to 5% and them wash for 5-10 minutes in tap water

12 . treated for 5 minutes with acidulous water (distilled water , 100 ml acetic acid 0.5 ml)

13 . perform the ascending scale of alcohols , xylene and close the slides in resin Eukitt .

best regards

Fabio Nobili

Dear Erhan Sahin

You can use the method of Mrasland et al. modification of Bielschowsky's sliver stain for nerve cells, axons and dendrites. I tried the paraffin sections and i got satisfied results. The steps are as follows:

Fixation:

Formol saline

Preparation of ammoniacal sliver solution:

To 30 ml of 20% sliver nitrate add 20 ml of absolute alcohol and mix; add strong ammonia (sp. gr. 0.880) drop by drop untile the precipitate first formed is just dissolved, then add a further five drops of strong ammonia.

The method is described in theory and practice of histological techniques by Bancroft and Stevens. Also, in cellular pathology technique by Culling, Allison and Barr.

Method:

1. Remove wax with xylene (2 changes each 5 minutes)

2. Replace xylene by absolute alcohol (2 changes each 5 minutes)

3. Dip into 0.5 - 1% cellodin for 20-30 seconds. Remove slide and allow excess celloidin to drain off. Wipe off excess celloidin from the back of the slide.

4. Flood the slide with 70% alcohol for 1 - 5 minutes to allow the celloidin film to harden.

5. Wash well in distilled water.

6. Place in 20% sliver nitrate solution for 25- 30 minutes (sometimes 25 - 60 minutes).

7. Wash in 10% formalin in tap water for 10-15 seconds. Sections should be pale yellow to brown.

8. Flood section with ammoniacal sliver nitrate solution and leave for 30 seconds.

9. Drain off sliver solution and flood with 10% formalin for 1-2 minutes. Examine under the microscope for proper staining and if necessary repeate steps 7 and 8.

10. Wash in distilled water.

11. Fix in 5% sodium thiosulfate for 1 - 5 minutes.

12. wash in tap or distilled water, dehydrate by ascending grades of alcohol, clear in xylene, and mount in DPX.

Best regards

Thanks alot. You are all so kind.

You can use this protocol,

J Neurosci Methods. 2006 May 15;153(1):135-46. Epub 2005 Nov 28.

A modified technique for high-resolution staining of myelin.

Pistorio AL1, Hendry SH, Wang X.